We propose a novel electro-stimulatory approach for drug and gene delivery involving stimuli at both the macro- and nano-scales. This approach will provide improved cell viability over traditional electroporation and be widely applicable in the medical sciences. It will also lay the groundwork for a unique approach to direct differentiation of patient-derived stem cells in vitro and provide the applicant with the background and resources necessary to lead future investigations in gene and drug delivery, regenerative medicine, and other areas of medical science where the use of electrical energy may be exploited. We believe that the co-localization of cationic peptides with the plasma membrane (PM) of cells will allow us to reduce and explicitly control the electric field strength required for electroporation. We hypothesize that co-localization of cationic peptides with the PM of cells will add to the transmembrane voltage required to electroporate the PM, thus reducing the required intensity of an externally applied electric field. By varying charge density and concentration of cationic peptides about the PM, we will be able to explicitly control the electroporative field threshold. The research will proceed in three stages. First, we will develop cationic peptides of various positive charge densities and characterize their short- and long-term toxicity as well their influence on stem cell multi-potency (aim 1). We will then determine the effect of cationic peptide presence and charge density on electroporative efficiency using a fluorescent plasma membrane integrity indicator (aim 2). Finally, we will characterize the relationship between cationic peptide charge density, peptide extracellular concentration and the electric field strength required for effective electrotransfection of a reporter gene (aim 3). While this technology has wide application possibilities in drug and gene delivery, we intend to implement this technique in regenerative medicine. If our results confirm our hypothesis, then ultimately we envision implementing the proposed technique in peptide-patterned polyethylene glycol) (PEG) hydrogels in order to provide both spatial and sequential control over genetic expression and cell differentiation in environments appropriate for tissue regeneration. Upon completion of this research, we will have developed a powerful tool for directing the complex sequence of events required in regenerating heterogeneous, functional tissues from patient-derived stem cells. Such engineered tissues will solve many of the problems associated with transplant therapy, including, but not limited to, tissue and organ availability and immunorejection. Transplantation of regenerated tissues will directly contribute to public health in treating burn victims, broken bones, blindness, deafness, heart and vascular damage, liver and kidney damage, and organ failure.